Biochemical and kinetic characterization of the recombinant GH28 Stereum purpureum endopolygalacturonase and its biotechnological application.

Endopolygalacturonase (EndoPG) from Stereum purpureum was expressed as a soluble protein in Pichia pastoris GS115, where after 3 days methanol induction the enzyme activity in the culture supernatant was 40 U mL-1. After purification by IMAC, SDS-PAGE analysis showed that the molecular weight of EndoPG was approximately 60.0 kDa. The carbohydrate content of the recombinant enzyme was estimated to be 67.0% (w/w). The optimum temperature and pH of catalysis were 60-70 °C and pH of 4.5, respectively. The enzyme was highly stable over the pH range 6.0-8.0 and retained approximately 60% of its initial activity after incubation at 70 °C for 30 min. The enzyme showed a specific activity of 5040.0 ± 217 U mg-1 and hydrolyzed citrus pectin with Vmax and a KM of 4947.10 ± 393.63 U mg-1 and 2.45 ± 0.23 mg mL-1, respectively, and showed a catalytic efficiency of 2052.90 ± 193.54 mL mg-1 s-1. EndoPG alone reduced the viscosity of papaya juice by 20% after 30 min, and increased its transmittance about 50% with a concomitant reduction of the color by about 55% after 5 h of enzymatic treatment. For apple juice, the relative reduction of viscosity was 30% after 5 h, and the reduction of the color was 30% with a 12% increase in transmittance. Supplementation of a commercial enzymatic cocktail for lignocellulose saccharification with EndoPG increased total reducing sugar release by 8.6 ± 2.1% against sugar cane bagasse, indicating improved access of the cellulolytic enzymes to the biomass polysaccharides.